Abstract
Purpose :
PEDF is an endogenous inhibitor of angiogenesis. Little is known about its effect on retinal endothelial cell (EC) function. Here we determined how deficiency in PEDF expression affects retinal EC proangiogenic activities.
Methods :
Retinal EC were prepared from wild type (PEDF+/+) and PEDF-deficient (PEDF-/-) mice. The expression of various EC markers and cell adhesion receptors were determined by FACS analysis. Cell adhesion to various extracellular matrix (ECM) proteins and cell migration using scratch wound and transwell migration assays were performed. The rate of cell proliferation was determined by counting the number of cells and DNA labeling. The level of oxidative stress was determined by dihydroethidium staining. Expression of various cell junctional and ECM proteins was determined by Western blot analysis. The rate of apoptosis was determined use caspase3/7 activity assay. Capillary morphogenesis was assessed in Matrigel. The level of vascular endothelial cell growth factor was determined using and ELISA assay.
Results :
The identity of retinal EC was confirmed by staining for EC markers VE-cadherin, CD31, and B4-lectin. Retinal EC expressed significant amounts of VEGF-R1 and endoglin, as well as ICAM-1, ICAM-2, and VCAM-1. PEDF-/- retinal EC proliferated at a faster and were less apoptotic under oxidative state challenge. Although the PEDF-/- retinal EC were less migratory in wound assay, they showed enhanced migration in a transwell assay compared with PEDF+/+ EC. PEDF-/- retinal EC were also less adherent and expressed increased levels of tenascin C, fibronectin, and collagen IV, while they produced lower amounts of osteopontin and thrombospondin-1. We also observed alterations in expression of number of integrins including α5, α3, and αv. PEDF-/- EC also exhibited reduced levels of CD31 and ZO-1, but increased levels of Occludin. PEDF -/- EC also failed to undergo capillary morphogenesis in Matrigel, exhibited increased levels of oxidative stress, and produced lower levels of VEGF compared to PEDF+/+ EC.
Conclusions :
PEDF expression has a significant impact on retinal EC adhesion and migration, perhaps through altered production of ECM and junctional proteins impacting their proangiogenic activity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.